The goal of this proposal is to gain a biophysical understanding of human orexin receptor agonist binding and activation, and to use this knowledge to develop small-molecule orexin receptor agonists as neuropharmacological tools and potential therapeutics for narcolepsy/cataplexy and other diseases. Narcolepsy is a life-long debilitating disorder affecting approximately 200,000 Americans, which is characterized by an inability to maintain wakefulness, sleep attacks, sudden loss of muscle function, and sleep paralysis. Current treatments for narcolepsy (such as psychostimulant drugs) do not treat the underlying neurochemical deficits and exhibit undesirable side-effects. Animal models and clinical investigations of human patients show that narcolepsy is caused by deficiency of the orexin (hypocretin) neuropeptides produced by neurons of the lateral hypothalamus, and that exogenous replacement of orexin activity may cure the disease. However, orexins cannot be used as therapeutic agents because they are peptides, which do not penetrate the blood-brain barrier and show poor activity after oral administration due to metabolic decomposition. We propose to use new technical advances in GPCR structural biology to determine X-ray structures of the orexin receptors in orexin-bound and small-molecule agonist-bound states, revealing the detailed non- covalent interactions that stabilize these complexes as well as changes in conformation of the receptors that are a consequence of agonist binding. In the second Aim, we will develop conformation-specific nanobodies that bind and stabilize the orexin receptor active state, and solve nanobody co-crystal structures to understand the propagated structural changes across the membrane that link the extracellular neuropeptide binding site and the intracellular G protein coupling site. In the third Aim, we will integrate structural insights with computational docking/simulation and medicinal chemistry to improve the affinity and potency of small- molecule orexin receptor agonists that were previously identified in a high-throughput screen. Our combination of strengths in GPCR structural biology, synthetic and medicinal chemistry, and computational chemistry places us in a unique position to design small-molecule orexin mimics with drug-like properties that can be further developed into therapeutics for the treatment of narcolepsy and other neurological disorders.